TY - JOUR
T1 - Construction and characterization of novel dual stress-responsive bacterial biosensors
AU - Mitchell, Robert J.
AU - Gu, Man Bock
N1 - Funding Information:
The authors would like to thank Dr. Marincs for pLITE201, Dr. Thomson for pMECA and Dr. Robert LaRossa for pDEW201, p KatG::LuxCDABE and p RecA::LuxCDABE , which were used in this study. We would also like to thank New England Biolabs for providing pACYC184. Robert would also like to thank the Lord Jesus for allowing him to do what he enjoys. This research was supported by the National Research Laboratory (2001 NRL) program of the Korea Institute of Science and Technology Evaluation and Planning (Project No. M10104000094-01J000004100). We, the authors, are grateful for the support.
PY - 2004/4/15
Y1 - 2004/4/15
N2 - Using the genes for the green fluorescence protein and Xenorhabdus luminescens luciferase operon and the promoters for the recA and katG genes, two stress-responsive Escherichia coli biosensor strains have been constructed that can individually or concurrently respond to oxidative and genotoxic conditions. Strain DUO-1 carries the pRGDK1 plasmid, which has the recA::GFPuv4 and katG::luxCDABE fusion genes oriented divergently with each other, while in DUO-2, i.e., pRGDK2, they are in a tandem orientation, with the recA promoter showing run-though transcription of the katG::luxCDABE fusion. These two strains and their responses were characterized using several known hydroxyl radical-forming chemicals, e.g., hydrogen peroxide and cadmium chloride, along with some genotoxins, e.g., mitomycin C and methyl-N-nitro-N-nitrosoguanidine, and some general toxicants. Both strains showed an induction of green fluorescent protein (GFP) and bioluminescence when they experienced DNA and oxidative damage, respectively, while the tandem orientation of the two fusion genes within DUO-2 allowed it to also sensitively respond to genotoxins via the production of bioluminescence. However, the characteristics of DUO-2's bioluminescent response to each stress were easily distinguishable, making it useful for the detection of both stresses. Furthermore, tests with mixtures of chemicals showed that both DUO-1 and DUO-2 were responsive when chemicals causing oxidative or genotoxic stress were present as a single chemical or within complex chemical mixtures.
AB - Using the genes for the green fluorescence protein and Xenorhabdus luminescens luciferase operon and the promoters for the recA and katG genes, two stress-responsive Escherichia coli biosensor strains have been constructed that can individually or concurrently respond to oxidative and genotoxic conditions. Strain DUO-1 carries the pRGDK1 plasmid, which has the recA::GFPuv4 and katG::luxCDABE fusion genes oriented divergently with each other, while in DUO-2, i.e., pRGDK2, they are in a tandem orientation, with the recA promoter showing run-though transcription of the katG::luxCDABE fusion. These two strains and their responses were characterized using several known hydroxyl radical-forming chemicals, e.g., hydrogen peroxide and cadmium chloride, along with some genotoxins, e.g., mitomycin C and methyl-N-nitro-N-nitrosoguanidine, and some general toxicants. Both strains showed an induction of green fluorescent protein (GFP) and bioluminescence when they experienced DNA and oxidative damage, respectively, while the tandem orientation of the two fusion genes within DUO-2 allowed it to also sensitively respond to genotoxins via the production of bioluminescence. However, the characteristics of DUO-2's bioluminescent response to each stress were easily distinguishable, making it useful for the detection of both stresses. Furthermore, tests with mixtures of chemicals showed that both DUO-1 and DUO-2 were responsive when chemicals causing oxidative or genotoxic stress were present as a single chemical or within complex chemical mixtures.
KW - Bacterial biosensor
KW - Bioluminescence
KW - Genotoxin
KW - Green fluorescent protein
KW - Oxidative damage
KW - Stress
UR - http://www.scopus.com/inward/record.url?scp=1342285620&partnerID=8YFLogxK
U2 - 10.1016/j.bios.2003.09.002
DO - 10.1016/j.bios.2003.09.002
M3 - Article
C2 - 15018952
AN - SCOPUS:1342285620
SN - 0956-5663
VL - 19
SP - 977
EP - 985
JO - Biosensors and Bioelectronics
JF - Biosensors and Bioelectronics
IS - 9
ER -